Toxic and bloom-forming Baltic Sea cyanobacteria under changing environmental conditions

The Baltic Sea is a shallow brackish water ecosystem. It is naturally prone to eutrophication, and massive cyanobacterial blooms are an annual phenomenon in this region. Blooms are toxic to humans and animals, and cause economical losses and harm for recreational users. Almost all cyanobacteria are photoautotrophic organisms, and many bloom-forming Baltic Sea cyanobacteria can additionally fix atmospheric nitrogen. Inorganic phosphorus is usually the first and most important growth-limiting factor. Monitoring the external phosphorus inflow is strictly implemented by the coastal states, but uneven point load still occurs. In addition, a heavy and long-term phosphorus load has resulted in substantial phosphorus reservoirs in the sediments and phosphorus can be circulated back to the waterbody. Cyanobacterial blooms in the Baltic Sea are dominated by Aphanizomenon sp., Dolichospermum sp., and Nodularia spumigena, of which Dolichospermum sp. and N. spumigena can produce toxins. Due to their evolutionary history, Dolichospermum sp. is more abundant in the less saline coastal regions, whereas N. spumigena dominates in the open sea. This work studied the effects of changing environmental conditions on the distribution and niche adaptation strategies of toxic and bloom-forming Baltic Sea cyanobacteria using state-of-the-art sequencing and molecular biology methods. Climate change models have predicted that the salinity of the Baltic Sea will possibly decline in the future, and thus the behavior of Dolichospermum sp. and N. spumigena was studied in unfavorable salinities. Comparative genomic analysis showed that Dolichospermum sp. has high synteny between its freshwater counterparts, and possibly therefore the strain was unable to proliferate in moderate salinities. Salt addition induced massive transcriptional shifts, especially within the photosynthesis and nitrogen-fixing pathways. Moreover, moderate salinity increased the production of microcystins and triggered the oxidative stress response. On the contrary, N. spumigena thrived in higher salinities, and its growth and metabolism were hindered by freshwater. Unique sigma factors and an elevated number of transposases were identified in the genome of N. spumigena, suggesting a high genetic capacity to adapt to changing salinities and brackish water conditions. The growth and metabolism of Dolichospermum sp. and N. spumigena were arrested under limited availability of inorganic phosphorus. Both strains upregulated the genes in the pho regulon, indicating that these genes can be used for determining the phosphorus status of cyanobacterial blooms. All studied strains of N. spumigena from the Baltic Sea carried the phnC-M gene cluster, which is responsible for the transport and utilization of the highly stable phosphonates. Naturally produced phosphonates were an additional phosphorus source for N. spumigena cyanobacteria, and produced a competitive advantage in phosphorus-limited conditions. However, methane, an organic remnant of methylphosphonate was released to the gaseous environment. Blooms of N. spumigena cyanobacteria may thus explain the elevated summertime methane concentration in the Baltic Sea. The results presented in this thesis suggest that cyanobacterial blooms will continue to appear in the future if sufficient amount of phosphorus is present but community composition may shift towards freshwater species as a consequence of climate change.Itämeri on matala murtovesiallas ja se on luontaisesti erittäin altis rehevöitymiselle. Massiiviset syanobakteeri- eli sinileväkukinnat ovatkin alueella jokakesäinen ilmiö. Kukinnat ovat myrkyllisiä ihmiselle ja muille nisäkkäille ja ne aiheuttavat haittaa veden virkistyskäyttäjille. Kaikki syanobakteerit ovat yhteyttäviä organismeja. Lisäksi Itämerellä kukintoja muodostavat lajit kykenevät sitomaan ilmakehän typpeä, minkä seurauksena epäorgaanisen fosforin saatavuus toimii usein syanobakteerien kasvua rajoittavana tekijänä. Itämerta ympäröivät valtiot valvovatkin nykyään tiukasti maalta tulevia epäorgaanisia fosforipäästöjä, mutta satunnaisia pistekuormituksia tapahtuu edelleen. Pitkään jatkunut raskas fosforikuorma on kuitenkin yhä sitoutuneena pohjan sedimentteihin, mistä se vapautuu vähitellen takaisin vesipatsaaseen. Itämeren syanobakteerikukinnat koostuvat Aphanizomenon sp., Dolichospermum sp. ja Nodularia spumigena -lajeista, joista kaksi jälkimmäistä tuottavat myrkkyjä. Evolutiivisen historiansa vuoksi Dolichospermum sp. esiintyy runsaammin vähäsuolaisemmilla rannikkovesialueilla, kun taas N. spumigena vallitsee suolaisemmalla avomerellä. Tässä työssä Itämeren myrkyllisten ja kukintoja muodostavien syanobakteerien esiintymistä ja kykyä sopeutua ilmastonmuutoksesta ja ihmisen toiminnan seurauksesta aiheutuvaan muuttuvaan ympäristöön on arvioitu nykyaikaisten sekvensointi- ja molekyylibiologisten työkalujen avulla. Koska Itämeren suolapitoisuuden on ennustettu madaltuvan ilmastonmuutoksen seurauksena, tämän työn ensimmäisessä osassa selvitettiin, miten Dolichospermum sp. ja N. spumigena käyttäytyvät epäsuotuisissa suolapitoisuuksissa. Laboratoriokokeet osoittivat, että Dolichospermum sp. on geneettisiltä ominaisuuksiltaan hyvin samankaltainen makean veden sukulaistensa kanssa, eikä se kykene kasvamaan korkeissa suolapitoisuuksissa. Korkeat suolapitoisuudet aiheuttivat mittavaa transkriptomin uudelleenjärjestäytymistä, mikä näkyi erityisesti fotosynteesi- ja typensidontakoneistojen toiminnan tukahduttamisena. Lisäksi korkea suolapitoisuus lisäsi myrkyntuotantoa sekä lipidien uudelleenjärjestäytymistä. N. spumigena kasvoi parhaiten korkeammissa suolapitoisuuksissa, ja matalammat suolapitoisuudet aiheuttivat metabolian hidastumista. N. spumigena -syanobakteerin genomisekvenssistä pystyttiin tunnistamaan ainutlaatuisia ominaisuuksia, joilla on todennäköisesti tärkeä rooli murtovesissä. Tutkittujen lajien kasvu ja metabolia hidastuivat, kun fosforin määrä oli rajoitettu. Molemmat lajit ilmensivät myös voimakkaasti pho-säätelyalueen geenejä, joita voidaan hyödyntää määritettäessä fosforin määrää syanobakteerikukinnoissa. Kaikilta tutkituilta Itämeren N. spumigena -kannoilta löydettiin phnC-M-geenialue, joka on vastuussa stabiilien fosfonaattimolekyylien kuljettamisesta ja pilkkomisesta. Työssä havaittiin, että luonnollisesti tuotetut fosfonaatit toimivat vaihtoehtoisena fosforinlähteenä N. spumigena -syanobakteereille ja tarjoavat lajille kilpailuedun alueilla, joilla epäorgaanisen fosforin saatavuus on niukkaa. N. spumigena ei kuitenkaan hyödyntänyt metyylifosfonaatista vapautuvaa orgaanista hiilimolekyyliä, metaania, ja metaanipitoisuuden määrä ympäröivässä kaasutilassa kasvoi. Massiivisilla N. spumigena -kukinnoilla saattaakin olla yhteys Itämeressä kesäisin havaittuihin kohonneisiin metaanipitoisuuksiin. Tulosten pohjalta voidaan päätellä, että syanobakteerikukinnat eivät katoa Itämerestä mikäli kokonaisfosforin määrää ei pienene, mutta kukintojen lajikoostumus saattaa muuttua ilmastonmuutoksen seurauksena

Changes in proteome and variation of the amount of the bioactive compounds of Anabaena sp. 90 while growing in different phosphorus media

Syanobakteerit ovat yhteyttäviä organismeja. Niitä tavataan tyypillisesti makeissa ja suolaisissa vesissä mutta esimerkiksi sienten kanssa symbioottisessa suhteessa eläviä lajeja on löydetty myös maalta. Epäorgaaninen fosfori (Pi) mielletään yleensä syanobakteerien kasvua rajoittavaksi tekijäksi vesiympäristöissä, sillä syanobakteerit voivat hyödyntää suoraan ainoastaan Pi:a fosforin lähteenä. Syanobakteereilta on löydetty Escherichia coli –bakteereilla tavattavia fosforin kuljetukseen osallistuvia pho-säätelyalueita ja niiden toimintaa on tutkittu jonkin verran. Syanobakteerit tuottavat yleensä lukuisia bioaktiivisia yhdisteitä, jotka voivat toimia esimerkiksi myrkkyinä tai estää bakteerien ja sienten kasvua. Bioaktiivisten yhdisteiden merkitystä syanobakteereille ei kuitenkaan tunneta. Suuri osa bioaktiivisista yhdisteistä tuotetaan ei-ribosomaalisella peptidisynteesillä (NRPS). Työn tavoitteena oli selvittää Anabaena 90 –syanobakteerikannan proteomissa tapahtuvia muutoksia ja bioaktiivisten yhdisteiden määrän vaihtelua, kun kantaa kasvatettiin matalassa (0,05 mg/l) ja korkeassa (5,5 mg/l) fosforipitoisuudessa. Ennen siirtoa kahdelle eri kasvatusalustalle solujen fosforivarastot tyhjennettiin kasvattamalla niitä fosforittomalla alustalla. Työssä käytettiin proteomien tutkimiseen kahdensuuntaista erottelevaa geelielektroforeesia (2D-DIGE) yhdistettynä LC/MS-analyyseihin. Bioaktiivisten yhdisteiden määrät analysoitiin myös LC/MS-ajoissa. Työssä käytettiin Anabaena 90 –syanobakteerikantaa. Kanta valittiin, koska sen genomi on kokonaan sekvensoitu ja annotoitu. Lisäksi kyseinen kanta tuottaa bioaktiivisia yhdisteitä: mikrokystiinejä, anabaenopeptiinejä ja anabaenopeptilidejä. Geeleiltä tunnistettiin yhteensä 11 proteiinia, joiden määrien todettiin tilastollisesti merkitsevästi kohonneiksi tai laskeneiksi matalassa fosforipitoisuudessa. Proteiineista kymmenen osoitettiin osallistuvan tiukkaan säätelyyn, joka aktivoituu, kun solukasvusto on saavuttamassa stationaarisen kasvun vaiheen. Tiukan säätelyn proteiinit osallistuivat aminohappojen metaboliaan sekä translaatioon. Yhden proteiineista osoitettiin olevan ribosomin pienen alayksikön proteiini. Lisäksi tunnistettujen proteiinien joukossa oli hiilen kiertoon osallistuva ribuloosibisfosfaatti-karboksylaasi/oksygenaasi (RuBisCO), jonka määrä väheni merkitsevästi matalassa fosforipitoisuudessa. Anabaena 90 –syanobakteerikannan bioaktiivisten yhdisteiden tuotannossa ei havaittu tilastollisesti merkitseviä eroja matalassa ja korkeassa fosforipitoisuudessa. Rinnakkaisten näytteiden määrää tulee lisätä, kun analyysi toistetaanCyanobacteria are phototrophic organisms. They usually occur in water but many species also live in terrestrial habitats, e.g. in symbiotic relationships with fungus. Inorganic phosphorus (Pi) is usually considered to be a limiting factor for the growth of cyanobacteria living in water, since cyanobacteria can use only Pi as a direct source of phosphorus. It has been shown that cyanobacteria have pho-regulon similar to that of Escherichia coli. Pho-regulon can transport and assimilate inorganic phosphate. Cyanobacteria usually produce a wide range of bioactive compounds, which can e.g. be toxic or prevent growth of other bacteria, fungi or yeast. Many of these compounds are produced by non-ribosomal peptide synthetases (NRPS). The aim of this study was to investigate changes in Anabaena sp. 90 proteome, and differences in amounts of bioactive compounds produced by the strain, while growing it in media with high (5,5 mg/l) or low (0,05 mg/ml) Pi concentration. Before moving the culture into two different Pi media, phosphorus storages of the culture were emptied by growing the strain in the media without Pi. In this study, 2D differential gel electrophoresis (2D-DIGE) coupled with LC/MS was used to study proteomes of the organism. Bioactive compounds were also analyzed by LC/MS. Anabaena sp. 90 was chosen because of its fully sequenced and annotated genome. The strain has been found to produce microcystins, anabaenopeptins and anabaenopeptilides. Eleven protein spots with significantly increased or decreased protein quantities were identified in the low Pi media. Ten of them were identified as proteins, which participate in bacterial stringent response. Stringent response is activated when culture is achieving the stationary phase. These stringent response proteins participate in the amino-acid metabolism and translation. One of the proteins was shown to be a ribosomal protein. In addition, the identified proteins included ribulose-bisphosphate carboxylase oxygenase (RuBisCO), which had a significantly lower concentration in the cells in low-phosphorous media. There were no significant differences in amounts of bioactive compounds when growing the culture in low and high Pi media. More replicates could be used, when the study of bioactive compounds is repeated

Cyanotoxins associated with macrophytes in Berlin (Germany) water bodies – Occurrence and risk assessment

Publisher Copyright: © 2022Fatal dog poisoning after uptake of neurotoxic cyanobacteria associated with aquatic macrophytes in Tegeler See (Berlin, Germany) raised concerns about critical exposure of humans, especially children, to cyanotoxins produced by macrophyte associated cyanobacteria during recreational activity. From 2017 to 2021 a total of 398 samples of macrophytes washed ashore at bathing sites located at 19 Berlin lakes were analysed for anatoxins, microcystins, and cylindrospermopsins, as were 463 water samples taken in direct proximity to macrophyte accumulations. Cyanotoxins were detected in 66 % of macrophyte samples and 50 % of water samples, with anatoxins being the most frequently detected toxin group in macrophyte samples (58 %) and cylindrospermopsins in water samples (41 %). Microcoleus sp. associated with the water moss Fontinalis antipyretica was identified as anatoxin producing cy-anobacterium in isolated strains as well as in field samples from Tegeler See. Anatoxin contents in macrophyte samples rarely exceeded 1 mu g/g macrophyte fresh weight and peaked at 9.2 mu g/g f.w. Based on established toxicological points of departure, a critical anatoxin content of macrophyte samples of 3 mu g/g f.w. is proposed. Five samples, all taken in Tegeler See and all associated with the water moss Fontinalis antipyretica, exceeded this value. Contents and concentra-tions of microcystins and cylindrospermopsins did not reach critical levels. The potential exposure risks to anatoxins for children and dogs are assessed and recommendations are given.Peer reviewe

Comparative Genomics of the Baltic Sea Toxic Cyanobacteria Nodularia spumigena UHCC 0039 and Its Response to Varying Salinity

Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spurn/germ UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.Peer reviewe

Divergent gene expression responses in two Baltic Sea heterotrophic model bacteria to dinoflagellate dissolved organic matter

Peer reviewe

Effects of allochthonous dissolved organic matter input on microbial composition and nitrogen cycling genes at two contrasting estuarine sites

Heterotrophic bacteria are important drivers of nitrogen (N) cycling and the processing of dissolved organic matter (DOM). Projected increases in precipitation will potentially cause increased loads of riverine DOM to the Baltic Sea and likely affect the composition and function of bacterioplankton communities. To investigate this, the effects of riverine DOM from two different catchment areas (agricultural and forest) on natural bacterioplankton assemblages from two contrasting sites in the Baltic Sea were examined. Two microcosm experiments were carried out, where the community composition (16S rRNA gene sequencing), the composition of a suite of N-cycling genes (metagenomics) and the abundance and transcription of ammonia monooxygenase (amoA) genes involved in nitrification (quantitative PCR) were investigated. The river water treatments evoked a significant response in bacterial growth, but the effects on overall community composition and the representation of N-cycling genes were limited. Instead, treatment effects were reflected in the prevalence of specific taxonomic families, specific N-related functions and in the transcription of amoA genes. The study suggests that bacterioplankton responses to changes in the DOM pool are constrained to part of the bacterial community, whereas most taxa remain relatively unaffected.Peer reviewe

Insight into the genome and brackish water adaptation strategies of toxic and bloom-forming Baltic Sea Dolichospermum sp. UHCC 0315

The Baltic Sea is a shallow basin of brackish water in which the spatial salinity gradient is one of the most important factors contributing to species distribution. The Baltic Sea is infamous for its annual cyanobacterial blooms comprised of Nodularia spumigena, Aphanizomenon spp., and Dolichospermum spp. that cause harm, especially for recreational users. To broaden our knowledge of the cyanobacterial adaptation strategies for brackish water environments, we sequenced the entire genome of Dolichospermum sp. UHCC 0315, a species occurring not only in freshwater environments but also in brackish water. Comparative genomics analyses revealed a close association with Dolichospermum sp. UHCC 0090 isolated from a lake in Finland. The genome closure of Dolichospermum sp. UHCC 0315 unraveled a mixture of two subtypes in the original culture, and subtypes exhibited distinct buoyancy phenotypes. Salinity less than 3 g L−1 NaCl enabled proper growth of Dolichospermum sp. UHCC 0315, whereas growth was arrested at moderate salinity (6 g L−1 NaCl). The concentrations of toxins, microcystins, increased at moderate salinity, whereas RNA sequencing data implied that Dolichospermum remodeled its primary metabolism in unfavorable high salinity. Based on our results, the predicted salinity decrease in the Baltic Sea may favor toxic blooms of Dolichospermum spp.Peer reviewe

Image8.PDF

<p>Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spumigena UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.</p

Table8.XLSX

<p>Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spumigena UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.</p

Image1.PDF

<p>Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spumigena UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.</p